In the past, antigen-specific lymphocytes have been detected by placing about 200,000 individual lymphocytes per well in a 96-well plate such as that shown in FIG. 3 and culturing them for from three days to a week (“Lymphocyte Function Detection Methods”, ed. by Junichi Yano, Michio Fujiwara, Chugai Igaku Corp. (1994) (Nonpatent Reference 1) and “Methods of Conducting Immunological Experiments I, II”, ed. by Shunsuke Ishida, Susumu Konda, Morosuke Moto, and Toshiyuki Hamaoka, Nankodo (1995) (Nonpatent Reference 2)).
These detection methods detect antigen-specific lymphocytes by:
1. Cell proliferation (uptake of 3H-thymidine, detection of live cells), and
2. Production of antibody and cytokine.
These methods are capable of determining the presence of antigen-specific lymphocytes in a lymphocyte population of about 200,000 cells. However, they are incapable of identifying individual antigen-specific lymphocytes present in the lymphocyte population.
By contrast, in recent years, a method of mixing antigen molecules labeled with fluorescent dye with lymphocytes to cause fluorescence-labeled antigen to bind to the antigen receptors of antigen-specific lymphocytes, and then using a flow cytometer to detect lymphocytes that have bound fluorescence-labeled antigen has been developed and put to practice (Altman, J. D., Moss, P. A., Goulder, P. J., Barouch, D. H., McHeyzer-Williams, M. G., Bell, J. I., McMichael, A. J., Davis, M. M., Phenotypic analysis of antigen-specific T lymphocytes, Science, 274: 94-96, 1996 (Nonpatent Reference 3)). This method is capable of identifying a single lymphocyte bound to antigen. It is also capable of separating out individual lymphocytes that bind antigen.
However, the above-cited method requires an expensive and complex device known as a cell sorter for separating out individual lymphocytes, and presents the following problems as well:
(1) It is difficult to set the separating conditions of the device, requiring skills for operating device to separate out cells;
(2) The background is high, precluding the detection of antigen-specific lymphocytes at frequencies of less than or equal to 0.1 percent;
(3) cell separation is inefficient;
(4) time is required to separate out cells of low frequency; and
(5) although antigen binding can be determined, it is difficult to analyze the reaction of the lymphocyte that has bound the antigen.
Another antigen-specific lymphocyte detection method has been developed in which antigen molecules bound to magnetic beads are mixed with lymphocytes to cause the magnetic bead-bound antigen to bind to the antigen receptors of the antigen-specific lymphocytes, and a magnet is then employed to separate the antigen-specific lymphocytes (Abts H., Emmerich M., Miltenyi S., Radbruch A., Tesch H. CD20 positive human B lymphocytes separated with the magnetic sorter (MACS) can be induced to proliferation and antibody secretion in vitro. Journal of Immunological Methods 125:19-28, 1989 (Nonpatent Reference 4)).
This method requires no complex device, cells are rapidly separated, and antigen binding can be determined. However, it is not possible to analyze the reaction of the lymphocyte in binding the antigen (the metabolic or physiological reaction of the cell, such as intracellular signal transduction, RNA synthesis, or protein synthesis). Further, the antigen-specific lymphocyte cannot be detected when the frequency of the antigen-specific lymphocyte is less than or equal to 0.1 percent.
Accordingly, the present invention has as its first object to provide a method of detecting antigen-specific lymphocytes that does not require a complex device, rapidly separates cells, permits the determination of antigen binding, permits the detection of antigen-specific lymphocytes (even at 0.001 percent and above), permits analysis of whether the lymphocyte that has bound the antigen reacts with antigen, and permits the separation of antigen-specific lymphocytes.
Further objects of the present invention are to provide a microwell array chip for detecting antigen-specific lymphocytes to be employed in the above-described detection method and a method of manufacturing antigen-specific lymphocytes using the above-described detection method.
The following are conventionally known methods of cloning antigen-specific antibody genes:
(1) In humans, there exists the method in which peripheral B lymphocytes are transformed with EB virus, antigen-specific antibody-producing cells are screened from the colonized cells, and antibody genes are cloned from the antigen-specific lymphocytes (Roome, A. J., Reading, C. L. The use of Epstein-Barr virus transformation for the production of human monoclonal antibodies. Exp. Biol. 43:35-55, 1984 (Nonpatent Reference 5), Carson, D. A., Freimark, B. D. Human lymphocyte hybridomas and monoclonal antibodies. Adv. Immunol. 38:275-311, 1986 (Nonpatent Reference 6)). This method is bothersome in that the screening of antigen-specific lymphocyte cell colonies is inefficient and a whole month is required to culture the cells. Although hybridomas can be produced for mice, no efficient human hybridoma system has yet been produced.
(2) A further method exists in which bacteriophage is employed to clone antigen-specific antibody genes (Huston, J. S., Levinson D., Mudgett-Hunter, M., Tai, M. S., Novotny, J., Margolies, M. N., Ridge, R. J., Bruccoleri, R. E., Haber, E., Crea, R., et al. Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia Coli. Proc. Natl. Acad. Sci., U.S.A. 85:5879-5883, 1988 (Nonpatent Reference 7)). In this case, mRNA is extracted from human lymphocytes, cDNA libraries consisting of the H and L chains of immunoglobulin, respectively, are prepared, the two are combined into single phage DNA, and the H and L chains are expressed by the phage. Antigen specificity is determined by the combination of H and L chains. However, in this system, the combinations are random and the phages producing antibody binding to the antigen are screened with antigen. As a result, when a phage producing antibody binding to antigen is produced, an antigen-specific antibody gene can be cloned. However, the random combination of H and L chains renders the screening of antigen-specific antibody genes highly inefficient. For example, assuming that the H chain cDNA and L chain cDNA of antibody for a given antigen are each present in the library at a frequency of one part in 104, the combination of an H chain and L chain capable of binding the antibody will be present at a frequency of one part in 108. Further, in this system, it is not known whether the combinations of H and L chains obtained are actually produced in the human body.
As set forth above, conventional methods of cloning antigen-specific antibody genes are highly inefficient. Even so, with considerable effort, it is possible to clone antigen-specific antibody genes by these methods. However, it is not possible to identify and select low-frequency antigen-specific lymphocytes using conventional methods.
Accordingly, the present invention has as its object to provide a method of conveniently selecting lymphocytes reacting with specificity to a certain antigen and efficiently cloning antigen-specific antigen receptor gene from the selected antigen-specific lymphocytes, both for antigen-specific lymphocytes of relatively high frequency and those of low frequency. Further objects of the present invention are to provide a method of manufacturing monoclonal antibody from the cloned antigen-specific immunoglobulin gene, and to provide a method of manufacturing materials for gene therapy using the cloned antigen-specific T-cell receptor gene.